The goal of this study is to understand how the corneal endothelial fluid pump works. The pump maintains corneal hydration and transparency. When the pump fails due to trauma, inflammation, ageing, or corneal dystrophy, corneal edema ensues, transparency is lost and vision is significantly degraded. The usual therapy is transplantation, which is not without significant compromises and complications. Knowing how the pump works is one approach to developing medical therapies that could delay or supplant the need for transplantation. As the population ages and the prevalence of endothelial dysfunction increases, demand for endothelial therapy will also increase. Ion transport is a key feature of the pump. Up to now the pump has been modeled as a classic ion secretory mechanism that is bicarbonate dependent and carbonic anhydrase sensitive. Previous studies and our work in the preceding period however, indicate that alternate models need to be investigated. Our overall hypothesis is that the corneal endothelium is a lactate removal pump. Because the cornea is very glycolytic and must remove the end product, which is lactic acid, we propose that the transcellular flux of lactate is coupled to water movement; tha lactate flux is via monocarboxylic acid transporters (MCTs); and that the buffering action of HCO3-, CA activity, membrane pHi regulators & HCO3- transporters act in concert to facilitate the flux of lactate. By buffering the protons transported by the MCTs, lactate:H+ & water flux is maximized by preventing reductions in the driving force for continued lactate:H+ transport. Using multiple in vitro & in vivo complementary approaches this will be tested in three aims. Aim 1 will investigate the role of buffering capacity on water and lactate fluxes. The flux ratio, i.e. mMoles lactate/ul water can be estimated to determine the tonicity of this osmotic coupling. If water coupled to lactate is significant, we expect isotonic (300 mEq/L) transport. Aim 2 will investigate the role of primary and secondary active transport (Na+,K+ ATPase,1Na+:2HCO3- cotransport, and Na+/H+ exchange) in facilitating lactate flux across the endothelium by determining the change in lactate flux ratio when these transporters are disturbed. Aim 3 will investigate the role of MCTs in facilitated lactate transport. Using pharmacological inhibitors and shRNA approaches in vivo we predict that inhibition of MCTs will have significant effects on corneal hydration. If the hypothesis is correct, we will have a more complete model of endothelial function that will allow further development of diagnostic and medical therapies or engineering of endothelial-like cells with the requisite transport properties. PUBLIC HEALTH RELEVANCE: The goal of this study is to understand how the corneal endothelial fluid pump works to keep the cornea transparent. Experiments are designed to test the hypothesis that the endothelium transports water from stroma to anterior chamber by facilitating the flux of lactic acid.